Methods for treating degenerative disc disease and chronic lower back pain

ABSTRACT

The present invention in various aspects and embodiments provides methods for the treatment or prevention of degenerative disc disease or chronic lower back pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US18/52539, which claims the benefit of U.S. Provisional PatentApplication Nos. 62/562,642, filed Sep. 25, 2017, and 62/572,027, filedOct. 13, 2017, the entire contents of which are herein incorporated byreference.

FIELD

The present invention relates to methods for treating infection as wellas the inflammatory state associated with degenerative disc disease andchronic lower back pain.

BACKGROUND

Chronic low back pain (CLBP) is a leading cause of disability andhospitalization. Although many conditions lead to CLBP, degenerativedisc disease (DDD) is among the most common diagnoses. Becausecorrelations among clinical symptoms, radiological signs of discdegeneration, and treatment outcomes are not satisfactory, a moredetailed understanding of the mechanisms of DDD and CLBP are needed toenable more effective treatment outcomes.

SUMMARY OF THE INVENTION

In various aspects and embodiments, the invention provides methods fortreating degenerative disc disease or chronic lower back pain. Themethod comprises administering to a patient having degenerative discdisease or chronic lower back pain, and suspected of having alow-virulence infection (such as a P. acnes infection), an antibioticand one or more of an IL-1β inhibitor and an inhibitor of Nerve GrowthFactor (NGF). In various embodiments, the method alleviates orameliorates the infective and inflammatory state that causes orexacerbates the patient's condition.

In some embodiments, the patient has chronic low back pain, and which isconsistent with structural intervertebral disc damage and/or consistentwith a low-virulence infection. Patients that have a low-virulenceinfection may be at increased risk of developing CLBP and/or may become“failed back surgery” patients, unless diagnosed correctly and treatedappropriately. In accordance with this disclosure, P. acnes is believedto significantly amplify etiological factors that contribute todegenerate disc disease (DDD) through the expression of virulencefactors and promotion of IL-1β and NGF, among others.

In some embodiments, the methods comprise detection or quantification ofPropionibacterium acnes (P. acnes) in patient samples, or othercommensal bacteria associated with low-virulence infection. In someembodiments, the patient has intervertebral disc disease or CLBP and afine needle biopsy is isolated for testing. In these embodiments, theinvention involves analysis of disc tissue for the presence of one ormore commensal pathogens (e.g., P. acnes) or associated virulencefactors or metabolites. In some embodiments, the presence of infectionis determined using disc tissue following discectomy.

In some embodiments, the invention involves detecting or quantifyingcommensal pathogen(s) (e.g., P acnes) in the disc tissue sample bymicrobiological cultivation and/or or by genetic, microbial-specificstain, immunochemical, or spectroscopic analysis. In some embodiments,the invention further comprises evaluating RNA from a disc tissue sample(e.g., mRNA or miRNA) to classify the profile as being indicative of lowvirulence infection, or not being indicative of a low virulenceinfection. In some embodiments, an RNA profile is evaluatedindependently to determine the presence of a low virulence infection,with or without the use of other techniques such as PCR, culture, ormicroscopy. In still other embodiments, the presence of thelow-virulence infection is determined in a non-invasive manner, forexample, by testing for an MR-spectroscopy signature. Non-invasivemethods obviate the need for biopsy or disc tissue samples.

In still other embodiments, treatment is provided post-surgery tofacilitate recovery and avoid recurrence. In such embodiments, disctissue following discectomy is available for determining the likelihoodthat the patient is suffering from a chronic infection of theintervertebral disc. In cases where a low-virulence infection is notconfirmed, the patient is not treated for a chronic infection.

Once infection has been established, therapeutic approaches combiningappropriate antibiotic treatment with a therapy or therapies targetingIL 10 and/or NGF and/or anti-angiogenic therapy (concomitantly orsequentially) are undertaken.

Antibiotics can be administered locally to the intervertebral discregion, or administered systemically. In some embodiments, antibiotics(alone or with other therapies) are applied locally during a biopsyprocedure. Exemplary antibiotics for P. acnes infection includeclindamycin and erythromycin. In some embodiments, the antibiotic is abeta-lactam antibiotic, macrolide, or tetracycline. In some embodiments,a beta-lactam antibiotic is administered with a beta-lactamase inhibitor(e.g., clavulanate). The patient in some embodiments receives chronicantibiotic therapy, for example, for at least about 1 month, or at leastabout 2 months, or at least about 3 months (e.g., about 100 days ormore).

In various embodiments, the patient further receives therapy with anIL-1β inhibitor. Exemplary IL-1β inhibitors include monoclonalantibodies against IL-1β antibody or fragment thereof, a recombinantprotein with IL-1β binding activity, or a small molecule inhibitor. Insome embodiments, the IL-1β inhibitor comprises a neutralizing orblocking antibody against IL-1β, a rIL-1RA, or an extracellular portionof the human IL-1R1 and/or IL-1 receptor accessory protein (IL-1RAcP).In still other embodiments, the IL-1β inhibitor is a caspase inhibitor(which prevents processing of proIL-1β),

In some embodiments, the patient receives therapy with an inhibitor ofNerve Growth Factor (NGF), which can be provided with antibiotictreatment, or in combination with antibiotic treatment and IL-1βinhibitor therapy. In some embodiments, the NGF inhibitor is selectedfrom a monoclonal anti-NGF antibody or fragment thereof, small-peptidemimetics of NGF, small-molecule TrkA antagonist, TrkA immunoadhesionmolecule, soluble binding domain of NGF receptor, or monoclonal antibodyor monoclonal antibody fragment against TrkA.

In some embodiments, the patient receives therapy with ananti-angiogenic therapy, such as a VEGF inhibitor, which can be providedwith antibiotic treatment, or in combination with antibiotic treatment,IL-1β inhibitor therapy, and/or NGF therapy. In some embodiments, theangiogenesis inhibitor is selected from a monoclonal anti-VEGF antibodyor fragment thereof, small-peptide mimetics, small-molecule antagonist(e.g., tyrosine kinase inhibitor), or soluble binding domain of VEGFreceptor. Various other anti-angiogenic treatments are well known.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use.

Embodiments of the invention will be further illustrated with thefollowing non-limiting examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a P. acnes-driven inflammatory state. P. acnes PAMPsbind to TLRs, which activate NF-κB, leading to the synthesis ofpro-IL-1β; P. acnes PAMPs also activate inflammasomes (NLRP3) andcaspase-1, which convert pro-IL-1β to IL-1β; P. acnes proteases directlyconvert pro-IL-1β to IL-1β.

FIG. 2 illustrates an IL-1β driven degenerative state. IL-1β stimulatesproduction of CC-chemokines that recruit myeloid cells, neurotrophinsNGF and BDNF that induce nerve ingrowth, and proteolytic enzymes (MMPs,ADAMTS4/5 aggrecanases) that degrade the ECM. Positive feedback loopsarise when IL-1β binds to IL-1R and when extracellular matrixdegradation products (DAMPs; e.g. fragmented collagen, aggrecan, andhyaluronic acid) activate TLRs. Both binding events further stimulateIL-1β production.

FIG. 3 illustrates a P. acnes driven, pain generating state. P. acnesdirectly activates nociceptors through several distinct mechanisms:N-formylated peptides activate FPR on a subset of nociceptor neuronsthat mediate mechanical hyperalgesia; CAMPs and pore-forming hemolyticfactors allow the entry of ions that lead to depolarization that inducesmechanical and thermal hyperalgesia.

FIG. 4 depicts relative lipase activity after 3 h and 24 h of 1:100 and1:1000 NP cells/P. acnes co-cultures, confirming viability of P. acnes.

FIG. 5 depicts average expression levels of pro-inflammatory cytokines(IL-113, IL-1α, IL-6, IL-8, CCL3, CCL4) at four time-points (3 h, 24 h,48 h and 1 week) and MOI100 and MOI1000 normalized to their levels innon-infected NP cells. LPS-treated cells were used as positive controls(*P<0.05, **P<0.01).

FIG. 6 illustrate an increase in the NGF and BDNF expression levels inP. acnes-infected NP cells after one week of infection (*P<0.05).

FIG. 7 shows an increase in IL-1β protein in NP cells after 24 hours ofP. acnes infection, as detected by ELISA (*P<0.05).

FIG. 8 depicts clindamycin suppression of the production of IL-1β, IL-6and IL-8 in P. acnes-infected NP cells. Averages from the experimentswith the three NP cell cultures are presented (*P<0.05, **P<0.01).

ABBREVIATIONS

ADAMTS-4/5, a disintegrin and metalloproteinase with thrombospondinmotifs 4/5; AF, anullus fibrosus; BDNF, brain-derived neurotrophicfactor; CAMP, Christie, Atkins and Munch-Petersen factors; CLBP, chroniclow back pain; DAMPs, damage-associated molecular patterns; DDD,degenerative disc disease; ECM, extracellular matrix; PAMPs,pathogen-associated molecular patterns; FPR, N-formylated peptidereceptor; IL-1R, interleukin-1 receptor; MMPs, matrixmetalloproteinases; NF-κB, nuclear factor KB; NGF, nerve growth factor;NP, nucleus pulposus; TLRs, Toll-like receptors, MOI, multiplicity ofinfection.

DETAILED DESCRIPTION

In various aspects and embodiments, the invention provides methods fortreating degenerative disc disease or chronic lower back pain. Themethod comprises administering to a patient having degenerative discdisease or chronic lower back pain, and suspected of having alow-virulence infection, an antibiotic and one or more of an IL-1βinhibitor, an inhibitor of Nerve Growth Factor (NGF), and ananti-angiogenic therapy. In various embodiments, the method alleviatesor ameliorates the infective and inflammatory state that causes orexacerbates the patient's condition. In various embodiments, the methodcan be employed with or without surgical interventions to resolve bothradicular pain and nociceptor pain that may be associated with thecondition.

As the term is used herein, a low-virulence infection is a chronic,low-grade, infection that is associated with a commensal microorganism.Exemplary commensal microorganisms include without limitation,Propionibacterium sp. (P. acnes) Staphylococcus sp. (e.g., coagulasenegative staphylococcus, or Staphylococcus aureus or Staphylococcusepidermidis), Corynebacterium, Lactobacillus sp., Pseudomonas sp. (e.g.,Pseudomonas aeruginosa), Enterococcus sp., Streptococcus sp. (e.g., S.pneumoniae), Bacillus sp. (e.g., Bacillus cereus), Citrobacter sp., E.coli, Moraxella sp., Haemophilus sp., Neisseria sp., Clostridium sp.,Enterobacter sp., and Klebsiella sp. In some embodiments, themicroorganism has a biofilm forming phenotype.

In some embodiments, the patient has chronic low back pain, and which isconsistent with structural intervertebral disc damage and/or alow-virulence infection. In some embodiments, the patient may be acandidate for, and may be scheduled for, intervertebral disc surgery.Patients that have a low-virulence infection may be at increased risk ofdeveloping CLBP and/or may become “failed back surgery” patients, unlessdiagnosed correctly and treated appropriately. For example, somepatients that undergo disc surgery will also suffer from CLBP prior tothe acute condition necessitating their surgery. Also, a certainproportion (around 5 to 10%) of patients undergoing disc surgery willdevelop CLBP in the follow-up, which are sometimes referred to as“failed back surgery” patients or “post-discectomy syndrome”. Theseconditions are statistically associated with a low-virulence infection.

Stirling et al. (2001) published the first evidence of bacterialinfection of the degenerated disc in 2001. Stirling et al. found that 19of 36 (53%) sciatica patients who had undergone microdiscectomy testedpositive for bacterial infection; 84% of these were Propionibacteriumacnes (P. acnes) infections. Subsequent research yielded conflictingresults, but two independent meta analyses (Urquhart 2015; Ganko 2015)reported the pooled prevalence of bacterial infection was 34% or 36.2%and P. acnes was identified as the major infecting species. They foundmoderate evidence that low virulent bacteria play a role in discdegeneration and moderate evidence of causation, but indicated thatthese observations could result from bacterial contamination. A morerecent 368-patient study (Capoor 2017) confirmed the previously observedprevalence and found that P. acnes was the only significant speciesisolated from degenerated disc tissue. Importantly, this studydocumented the presence of P. acnes biofilm in the infected disc tissue;a result consistent with infection rather than perioperativecontamination.

While antibiotic therapy has seen some moderate results in the treatmentof CLBP, in accordance with this disclosure, it is believed thatantibiotic therapy alone may not be effective to relieve the chronicinflammatory state, particularly of damaged intervertebral discs, whichis susceptible to recurrent infection by P. acnes. Further, somepatients will not have a low virulence infection, and thus should not beunnecessarily treated with antibiotics.

P. acnes plays a key role in the development of acne vulgaris, as wellas roles in other chronic and recurrent infections, including implantinfections, which are facilitated by the organism's ability to formbiofilms. In pilosebaceous units, P. acnes pathogen-associated molecularpatterns (PAMPs) bind to toll-like receptor (TLR) 2 on sebocytes. Thisactivates NF-κB signaling and results in the production of pro-IL-1β. P.acnes PAMPs also trigger NLRP3 inflammasome activation, whichfacilitates pro-IL-1β cleavage and the excessive release of matureIL-1β. IL-1β promotes dermal matrix destruction through induction ofmatrix metalloproteases (MMPs). Progression of the inflammation resultsin follicular rupture, allowing P. acnes to leak out of thepilosebaceous unit and activate perifollicular myeloid cells. Thisresults in further release of IL-1β and neutrophil-rich perifollicularinflammation. Thus, IL-1β is the driver of inflammatory responses to P.acnes in acne vulgaris.

Disc degeneration is known to be mediated by the abnormal secretion ofcytokines by the inner nucleus pulposus (NP) and outer annulus fibrosus(AF) cells of the intervertebral disc, as well as by immune cellsattracted to the site of disc degeneration. The initiating insult isthought to trigger NF-κB signaling through TLRs and stimulate theproduction of pro-IL-1β. Increased levels of NLRP3 and caspase-1 havealso been described in degenerated disc tissue, indicating inflammasomeactivation and facilitated maturation of pro-IL-1β. In addition, P.acnes proteases have been found to convert pro-IL-1β to mature IL-1β.Inflammatory actions are further amplified because IL-1β is not only anNF-κB target gene, but also an NF-κB activator, forming a positivefeedback loop. This IL-1β-IL-R1 signaling promotes extracellular matrixdegradation through induction of proteolytic enzymes, including MMPs 1,2, 3, 9, and 13 and aggrecanases of ‘a disintegrin and metalloproteinasewith thrombospondin motifs (ADAMTS) families 4/5. Specificdamage-associated molecular patterns (DAMPs), which include fragmentedcollagen, aggrecan, or hyaluronic acid, bind to TLRs on NP and AF cellsand activate NF-κB, forming another positive feedback loop. IL-1β alsopromotes the production of chemotactic CC-chemokines, mainly CCL3 andCCL4, leading to the recruitment and activation of infiltrating immunecells that further amplify the inflammatory cascade. These pathways formthe core of a model in which P. acnes infection causes IL-1β release byNP, AF and immune cells, which leads to extracellular matrix degradationwithin the intervertebral disc.

The ingrowth of nociceptive nerve fibers into the degenerated disc,usually accompanied by the presence of annular fissures can be a mainsource of nociception related to CLBP. IL-1β has been shown to inducethe expression of neurotrophin-like nerve growth factor (NGF) andbrain-derived neurotrophic factor (BDNF) in both disc and immune cells,supporting nerve ingrowth into the degenerated disc. IL-1β-stimulatedNGF and BDNF production further induces expression of pain-associatedcation channels in the dorsal root ganglion, the depolarization of whichis likely to promote low back and radicular pain. Finally, NGF has adirect activating or sensitizing effect on nociceptors, and itsupregulation in CLBP has been demonstrated.

Disc cells (NP and AF) make up a small portion of the disc tissue massand primarily remove waste and receive nutrition through diffusion. Indegenerative disc disease, there can be nerve ingrowth and likely somelevel of angiogenesis to support the nerve ingrowth. In accordance withthis disclosure, P. acnes produces alpha-hemolysins and other virulencefactors that interact with nociceptors in nerve in-growths, resulting inCLBP. Further, in accordance with this disclosure, P. acnes is believedto significantly amplify etiological factors that contribute todegenerate disc disease (DDD) through the promotion of IL-1β andfacilitates pathogenesis in synergy with other etiological factors ofDDD.

Since activation of IL-1β system promotes low back pain, P. acnes asextensive inducer of IL-1β indirectly participates in the pathogenesisof CLBP. Based on the ability of Staphylococcus aureus to activatesensory neurons by releasing formyl peptides and thepore-forming/hemolytic virulence factor α-haemolysin, it is believedthat there is also a direct, IL-1β independent, mechanism of P. acnesinvolvement in CLBP realized through the release of pore-forminghemolysins that induce calcium flux and action potentials in nociceptiveneurons. This is supported by the fact, that P. acnes genome containsseveral putative pore-forming/hemolytic virulence factors (e.g. genesPPA687, PPA1198, PPA1231, PPA1340, and PPA2108 encoding homologs ofChristie, Atkins and Munch-Petersen (CAMP) factors, potentiallyhemolytic gene products including PPA565, which shares similarity withhemolysin III of B. cereus, and PA938 (COG1253) and PPA1396 (COG1189),which share similarity with the hemolysins TlyC and TlyA of Brachyspirahyodysenteriae, respectively). These hemolytic factors are clinicallyrelevant, as hemolysis can be used as a clinical marker for the presenceof P. acnes in orthopedic infections. Therefore, P. acnes can play arole in the pathogenesis of CLBP, and P. acnes bacterial load could bedirectly linked to clinical course of CLBP.

In some embodiments, the methods comprise detection or quantification ofPropionibacterium acnes (P. acnes) or infection thereof in patientsamples, or infection by other commensal bacteria associated withlow-virulence infection. P. acnes is a gram-positive aerotolerantanaerobe that forms part of the normal resident microbiota of the skin,oral cavity and the gastrointestinal and genito-urinary tracts. It is anopportunistic pathogen that has been linked to a wide range ofinfections and conditions, including implant infections, discitis,musculoskeletal conditions (e.g., osteitis, osteomyelitis,synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome),sarcoidosis, chronic prostatitis, and prostate cancer.

Thus, in some embodiments, the patient has intervertebral disc diseaseor CLBP and a fine needle biopsy is isolated for testing. In theseembodiments, the invention involves analysis of disc tissue for thepresence of infection of one or more commensal pathogens (e.g., P.acnes), and/or for the presence of an RNA signature indicative of alow-virulence infection. In some embodiments, the presence of infectionis determined using disc tissue following discectomy.

In some embodiments, the invention involves detecting or quantifyingcommensal pathogen(s) (e.g., P acnes) in the disc tissue sample bymicrobiological cultivation or by genetic, microbial-specific stain,immunochemical, or spectroscopic analysis. In some embodiments, theinvention further comprises evaluating the RNA from a sample (e.g., mRNAor miRNA) to classify the profile as being indicative of low virulenceinfection, or not being indicative of a low virulence infection. In someembodiments, an RNA profile is evaluated independently to determine thepresence of a low virulence infection, with or without the use of othertechniques such as PCR, culture, or microscopy.

In some embodiments, an RNA profile (of host tissue) is evaluated forthe presence of an RNA signature (e.g., mRNA or miRNA signature) that isindicative of a low-grade or low-virulence infection. Exemplary RNAsignatures are disclosed in WO 2017/019440, which is hereby incorporatedby reference. An exemplary miRNA score for P. acnes infection can bedetermined by scoring the relative expression levels of miR-29a-3p andmiR-574-3p. For example, a diagnostic miRNA score (DMS) based on thefollowing formula results in high specificity using a cut-off of −0.3:DMS=18.71−11.24*log 10 (miR-29a-3p)+10.4*log 10 (miR-574-3p). Other RNAsignatures can be trained from RNA profiles of samples that are positiveor abundant for P. acnes (or other commensal microorganism) and samplesthat test negative (or non-abundant) for P. acnes (or other commensalmicroorganisms). For example, samples can be binned based on detection(or detection level) of P. acnes by quantitative PCR and at least oneother technique, such microbial culture or spectroscopy (including withmicrobial stains, FISH, or immunohistochemistry).

In some embodiments, the presence of a low-virulent infection isevaluated by culture, including aerobic and/or anaerobic cultivation andsubsequent biochemical and spectroscopic (e.g., mass spec., MALDI-TOFMS, or NMR) identification of species. For genetic analysis, nucleicacids (e.g., DNA and/or RNA) are isolated from the sample for analysis.In still other embodiments, immunochemistry (e.g., immunohistochemistryor ELISA) can be used to detect protein or other epitopes of commensalorganisms. For instance, without limitation, monoclonal antibodiesagainst P. acnes antigens including virulence factors can be used, suchas antibodies specific for epitopes of cell-membrane-bound lipoteichoicacid (PAB antibody) and ribosome-bound trigger factor protein (TIGantibody). In some embodiments, antibodies specific for P. acnesalpha-hemolysin or formylated peptide is used to identify thesevirulence factors in biopsy or disc material, which indicates that thepatient is suffering from a low virulence infection. Other techniquessuch as microscopy on tissue samples can be used to identify positivesamples, and may employ any appropriate staining reagent (e.g., gramstain, P. acnes-specific stain, or fluorescent in situ hybridization(FISH)) or other immunoreagents specific for the commensal microorganismof interest. Mass spectroscopy techniques can be used to identify P.acnes-specific molecules that are indicative of infection, includingbiofilm components.

In some embodiments, the presence or level of commensal pathogens isdetermined (alternatively or in addition to culturing) by hybridizationor amplification of microbial nucleic acids. For example, detectionassays include real-time or endpoint polymerase chain reaction (PCR),nucleic acid hybridization to microarrays, or nucleic acid sequencing.In some embodiments, the molecular detection assay detects microbialribosomal RNA (rRNA) genes, such as 16S and/or 23S rRNA genes, andparticularly the variable regions of 16S. In some embodiments, thepresence of a commensal pathogen is determined by a microarrayhybridization-based assay. Exemplary methods for diagnosing alow-virulence infection of an intervertebral disc are described in WO2017/019440, which is incorporated herein by reference.

In some embodiments, the presence of the low-virulence infection isdetermined in a non-invasive manner. For example, in some embodiments anMR-spectroscopy signature is obtained to determine the chemicalcomposition of the intervertebral disc. The chemical composition maycomprise biofilm components specific for P. acnes biofilm, as well asother molecules indicative of P. acnes infection. Exemplary methods forperforming MRS on intervertebral discs are described in US 2011/0087087,which is hereby incorporated by reference in its entirety.

In some embodiments of the present invention, where the diagnostic testconfirms a low-virulence infection, the patient is treated for infectionbefore any invasive surgery (such as discectomy), and in someembodiments, invasive procedures may be entirely avoided.

In still other embodiments, treatment is provided post-surgery tofacilitate recovery and avoid recurrence. In such embodiments, disctissue following discectomy is available for determining the likelihoodthat the patient is suffering from a chronic infection of theintervertebral disc. In cases where a low-virulence infection is notconfirmed, the patient is not treated for a chronic infection.

Antibiotic treatment consisting of amoxicillin (500 mg)-clavulanic acid(125 mg) three times a day for 100 days has been used in patients withCLBP unresponsive to conservative therapies with mixed results. [Albert2013; Palazzo 2017] However, the patient populations in these studieswere not evaluated for the presence of infection or IL-1β overexpressionprior to treatment; the treatment provided was not typical of theantibiotic regimens normally used to treat P. acnes infection (Jahns2016; Zeller 2007); and even if P. acnes infection were to besuccessfully eradicated, other etiological factors could support ongoingIL-1β-based inflammatory process and NGF-dependent nerve ingrowth andnociception, leading to treatment failure.

Once infection has been established, therapeutic approaches combiningappropriate antibiotic treatment with a therapy or therapies targetingIL-1β and/or NGF and/or angiogenesis (concomitantly or sequentially) areundertaken. A range of therapies targeting these factors are available,including several monoclonal antibody therapies. Development of anappropriate diagnostic strategy will rationalize indication of thecombination therapy and a provide path to precision medicine in thetreatment of DDD and CLBP.

Antibiotics can be administered locally to the intervertebral discregion (e.g., by injection or transdermally), or administeredsystemically (e.g., orally or i.v.). In some embodiments, antibiotics(alone or with other therapies) are applied locally during a biopsyprocedure. Exemplary antibiotics for P. acnes infection includeclindamycin and erythromycin. In some embodiments, the antibiotic is atetracycline antibiotic, such as tetracycline, minocycline, doxycycline,oxytetracycline and lymecycline. In various embodiments, depending onthe etiology of the infection, the antibiotic can be a beta-lactamantibiotic, macrolide, or tetracycline. For example, the antibiotic isselected from benzylpenicillin, amoxicillin, ampicillin, dicloxacillin,methicillin, nafcillin, oxacillin, penicillin G, cephalexin, cefoxitin,cephalolothin, ceftriaxone, ciprofloxacin, chloramphenicol,erythromycin, tetracycline, vancomycin, clindamycin, fusidic acid,doxycycline, moxifloxacin, linezolid, rifampicin, ertapenem,taurolidine, or a combination thereof. In some embodiments, abeta-lactam antibiotic (such as amoxicillin) is administered with abeta-lactamase inhibitor (e.g., clavulanate). The antibiotic givenorally are generally selected from those that are able to penetrate theintervertebral disc.

The patient in some embodiments receives chronic antibiotic therapy, forexample, for at least about 1 month, or at least about 2 months, or atleast about 3 months (e.g., about 100 days or more).

In various embodiments, the patient further receives therapy with anIL-1β inhibitor. Exemplary IL-1β inhibitors include monoclonalantibodies against IL-1β antibody or fragment thereof, a recombinantprotein with IL-1β binding activity, or a small molecule inhibitor.Exemplary antibody fragments include single chain variable fragments. Insome embodiments, the IL-1β inhibitor comprises a neutralizing orblocking antibody against IL-1β, a rIL-1RA, or an extracellular portionof the human IL-1R1 and/or IL-1 receptor accessory protein (IL-1RAcP),which can be linked in-line to IgG Fc region. In still otherembodiments, the IL-1β inhibitor is a caspase inhibitor (or otherinhibitor that prevents processing of proIL-1β), and which is optionallyadministered orally.

Exemplary IL-1β inhibitors are selected from anakinra, rilonacept,canakinumab, geviokizumab, LY2189102, MED-8968, CYT013, sIL-1RI,sIL-1RII, EBI-005, CMPX-1023, VX-765 (Pralnacasan), IL-1trap, CDP-484,CP424174, CP412245, CJ14877, CJ14897, and LL-Z1271a.

The IL-1β antagonist or inhibitor can be administered by a routeselected from parenteral, oral, and transdermal. Generally, where theinhibitor is a biologic, such as an antibody or portion thereof or otherrecombinant protein, the inhibitor may be administered parenterally,including subcutaneous injection, intramuscular injection, intravenousinjection, or local injection to intervertebral disc tissue. In someembodiments, the therapy is provided during a biopsy procedure. In someembodiments, the IL-1β inhibitor is a small molecule (such as caspaseinhibitor), and may be administered orally or transdermally in someembodiments.

In some embodiments, the therapy with the IL-1β inhibitor is concurrentwith antibiotic therapy (e.g., for at least about 1 month, 2 months, or3 months) or is administered after antibiotic therapy. In someembodiments, the course of therapy with the IL-1β inhibitor is shorteror longer than antibiotic therapy, such as about 2 weeks, about 3 weeks,about 1 month, about 2 months, about 3 months, or for 6 months or more.Administrations may be given daily, weekly, or every other week, ormonthly. Generally, biologics are administered weekly, every other week,or monthly. In some embodiments, the patient receives from 2 to 8 dosesof IL-1β inhibitor.

In some embodiments, the patient receives therapy with an inhibitor ofNerve Growth Factor (NGF), which can be provided with antibiotictreatment, or in combination with antibiotic treatment and IL-1βinhibitor therapy. In some embodiments, the NGF inhibitor is selectedfrom a monoclonal anti-NGF antibody or fragment thereof (including ascvf), small-peptide mimetics of NGF, small-molecule TrkA antagonist,TrkA immunoadhesion molecule, soluble binding domain of NGF receptor, ormonoclonal antibody or monoclonal antibody fragment against TrkA.Tropomyosin receptor kinase A (TrkA) is also known as high affinitynerve growth factor receptor. In some embodiments, the inhibitor of NGFis tanezumab, fulranumab, TrkAd5, AMG 403, Appha-D11, MNAC13, ALE0540,PD90780, or PPC-1807.

The NGF inhibitor can be administered by a route selected fromparenteral, oral, and transdermal, and in some embodiments isadministered during a biopsy procedure. Generally, where the inhibitoris a biologic, such as an antibody or portion thereof or otherrecombinant protein, the inhibitor may be administered parenterally,including subcutaneous injection, intramuscular injection, intravenousinjection, or local injection to intervertebral disc tissue. In someembodiments, the NGF inhibitor is a small molecule, and may beadministered orally or transdermally in some embodiments.

In some embodiments, the therapy with the NGF inhibitor is concurrentwith antibiotic therapy (e.g., for at least about 1 month, 2 months, or3 months) or is administered after antibiotic therapy. In someembodiments, the course of therapy with the NGF inhibitor is shorter orlonger than antibiotic therapy, such as about 2 weeks, about 3 weeks,about 1 month, about 2 months, about 3 months, or for 6 months or more.Administrations may be given daily, weekly, or every other week, ormonthly. Generally, biologics are administered weekly, every other week,or monthly. In some embodiments, the patient receives from 2 to 8 dosesof IL-1β inhibitor. The NGF inhibitor therapy may be concurrent withIL-1β therapy, including separate or co-formulation.

In some embodiments, the patient receives an anti-angiogenic therapy,which can be provided with antibiotic treatment, or in combination withantibiotic treatment and IL-1β inhibitor therapy and/or anti-NGFtherapy. In some embodiments, the angiogenesis inhibitor is a VEGFpathway inhibitor, which can be a monoclonal anti-VEGF antibody orfragment thereof (including a scvf), small-peptide mimetics,small-molecule inhibitor (e.g., tyrosine kinase inhibitor), or solublebinding domain of VEGF receptor. Exemplary angiogenesis inhibitorsinclude antibodies directed against VEGF or VEGFR, soluble VEGFR/VEGFRhybrids, and tyrosine kinase inhibitors. An exemplary VEGF pathwayinhibitor is Bevacizumab. Bevacizumab binds to VEGF and inhibits it frombinding to VEGF receptors.

The angiogenesis inhibitor can be administered by a route selected fromparenteral, oral, and transdermal, and in some embodiments isadministered during a biopsy procedure. Generally, where the inhibitoris a biologic, such as an antibody or portion thereof or otherrecombinant protein, the inhibitor may be administered parenterally,including subcutaneous injection, intramuscular injection, intravenousinjection, or local injection to intervertebral disc tissue. In someembodiments, the angiogenesis inhibitor is a small molecule, and may beadministered orally or transdermally in some embodiments.

In some embodiments, the therapy with the angiogenesis inhibitor isconcurrent with antibiotic therapy (e.g., for at least about 1 month, 2months, or 3 months) or is administered after antibiotic therapy. Insome embodiments, the course of therapy with the angiogenesis inhibitoris shorter or longer than antibiotic therapy, such as about 2 weeks,about 3 weeks, about 1 month, about 2 months, about 3 months, or for 6months or more. Administrations may be given daily, weekly, or everyother week, or monthly. Generally, biologics are administered weekly,every other week, or monthly. In some embodiments, In some embodiments,the patient receives from 2 to 8 doses of IL-1β inhibitor. Theangiogenesis inhibitor therapy may be concurrent with IL-1β therapy,including separate or co-formulation.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art. Formulation components suitable forparenteral administration include a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as EDTA; buffers such asacetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ or phosphate buffered saline(PBS). The carrier should be stable under the conditions of manufactureand storage, and should be preserved against microorganisms. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof.

Embodiments of the invention will be further illustrated with thefollowing non-limiting examples.

EXAMPLES Example 1. Ability of P. acnes to Induce IL-1β-BasedPathogenesis

The ability of P. acnes PAMPs to induce IL-1β and trigger NLRP3inflammasome assembly are demonstrated by monitoring of IL-1β, NLRP3 andcaspase 1 levels in ex vivo models based on NP and AF cell culturesderived from clinical specimens of degenerated disc tissue. In vivomodels in which animals are experimentally infected with P. acnesstrains isolated from human degenerated discs could also be employed.Further, short fragments of hyaluronic acid, P. acnes-induced DAMPs,shown to interact with TLR2 of the NP cells in vitro, could also beconfirmed to induce IL-1β in an animal experiment.

To further establish that P. acnes is a strong contributor toIL-1β-based pathogenesis of DDD, in vitro and in vivo experiments areperformed to quantitatively compare the ability of P. acnes to induceIL-1β with other known activators of IL-1β used in the experimentalstudies of DDD. Ability of the P. acnes related formyl peptides and thepore-forming/hemolytic virulence factor to induce calcium flux andaction potentials in nociceptive neurons could be evaluated in vitrousing analogical designs as used in study of Staphylococcus aureus. ¹⁹

Expression levels of pro-inflammatory cytokines and nerve growth factorswere assessed in P. acnes-infected cell cultures derived from humantissue samples of degenerated nucleus pulposus (NP).

Eight NP cell primary cultures were established from human tissuesamples of degenerated nucleus pulposus (NP) that were obtained fromeight patients who underwent microdiscectomy for lumbar disc herniation.Further, a P. acnes strain (type II) derived from P. acnes stronglypositive herniated disc tissue was used for in vitro infection of NPcells disc cultures.

Experiments were performed in triplicates with two multiplicities ofinfection (MOI), e.g., 100, 1000, and expression levels ofpro-inflammatory cytokines (e.g., IL-1β, IL-1α, IL-6, IL-8, CCL3, CCL4)and nerve growth factors (e.g., NGF, BDNF) were quantified byquantitative real-time PCR at four time-points (3 h, 24 h, 48 h and 1week). Non-infected cells were used as negative controls and LPS-treatedcells were used as positive controls. FIG. 4 depicts the presence ofviable P. acnes strain in the NP culture, as confirmed by P. acneslipase assay. Specifically, relative lipase activity of 1:100 and 1:1000NP cells/P. acnes co-cultures was assayed after 3 hours and 24 hours,confirming the viability of P. acnes. FIG. 5 shows the averageexpression levels of the tested genes: IL-1β, IL-1α, IL-6, IL-8, CCL3,and CCL4. The data shows significant increase in expression of alltested cytokines with the most significant changes observed after 24hours and MOI 1000.

On the other hand, when the expression changes of the cytokines wereevaluated at the level of the individual NP cell cultures, it wasobvious that the dynamics of the inflammatory response differed betweenthe cell cultures. For example, fold changes related to IL-1β presentedacross various cell lines and under different conditions are summarizedin Table 1 below:

TABLE 1 IL-1β expression changes in various cell lines and underdifferent conditions (+, fold-change > 1 < 2; ++, fold-change > 2 < 5;+++, fold-change > 5). MOI100 MOI1000 MOI100 MOI1000 MOI100 MOI1000MOI100 MOI1000 3 h 3 h 24 h 24 h 48 h 48 h 1 w 1 w KB7007 + ++ −+++ + + + − RT7806 + ++ + +++ − − − + MP7910 +++ +++ +++ +++ +++ +++ − +ML7364 +++ +++ + ++ + ++ − − SM7859 ++ ++ − + − ++ − − JP8161 − − − ++ −− ++ +++ VC5509 − − ++ + ++ +++ + ++ MH6257 − − +++ +++ − ++ − −

In some cases, an observed cytokine response did not present until after24 hours.

The data further showed that no significant changes were found in NGFand BDNF expression levels after 3 h, 24 h and 48 hours of lastinginfection. FIG. 6 depicts an MOI-dependent significant increase in NGFand BDNF expression levels in P. acnes-infected NP cells observed afterone week of infection. In the case of the disc degeneration driving thecytokine IL-1β, FIG. 7 shows that its protein expression levels werealso increased 24 hours post-infection, as detected by ELISA.

Since P. acnes is known to be sensitive to the antibiotic clindamycin inthe therapy of acne vulgaris, this study further sought to assess theeffect of clindamycin treatment in suppressing production and expressionof pro-inflammatory cytokines in human NP cells induced by P. acnes.

Accordingly, NP cell cultures derived from three specimens of clinicaldisc tissue were infected with one of the P. acnes strains (type II)derived from P. acnes strongly positive herniated disc tissues (MOI1000). Half of the plates were treated with 0.25 g/mL clindamycin, andexpression levels of IL-1β, IL-6 and IL-8 were measured after 24 hoursand 48 hours. Significantly lower expression levels of all cytokines atboth time points were observed, as shown in FIG. 8, indicating theability of clindamycin to suppresses expression of pro-inflammatorycytokines in human NP cells induced by P. acnes.

Materials and Methods

Microbiological Culture

The disc fragment for culture was weighed, placed into a Micro Bag(Seward) containing 4 ml of Viande-Levure medium, and homogenized with aStomacher 80 (Seward) under aseptic conditions. 100 μl of the resultanthomogenate was inoculated onto Wilkins Chalgren Anaerobic Agar with 7%sheep's blood and vitamin K (Hi Media Laboratories). An Anaerobic WorkStation Concept 400 (Ruskinn Technology) was utilized for culture;inoculated plates were incubated for 14 days at 37° C. with anatmosphere of 80% N2, 10% CO2, and 10% H2. The same amount of thehomogenate was also cultured aerobically on Columbia Blood Agar (Oxoid)for 7 days at 37° C. in order to detect aerobic bacteria. Followingincubation, the bacterial colonies were counted and the quantity of eachcolonial morphotype was expressed as colony forming units (CFU) per gramof tissue. In the case of P. acnes positivity, a single P. acnes colonywas taken and inoculated on a new anaerobic plate and incubated underanaerobic condition at 35-37° C. until colonies appeared. Frominoculated plate, full sterile loop was taken and placed in glycerolserum broth media in 2 mL sterile cryo tube and placed in −80° C.freezer.

Study Participants

Human tissue samples of degenerated Nucleus pulposus (NP) were obtainedfrom 8 patients who underwent microdiscectomy at the University HospitalBrno, Czech Republic.

Isolation and Culture of Human Nucleus Pulposus (NP) Cells

Fresh nucleus pulposus (NP) tissue samples were cut into small piecesusing a sterile, individually packaged, gamma-irradiated scalpel and, asterile, gamma-irradiated petri dish and then digested overnight withcollagenase A (Roche) at 37° C. After the digestion, the cellsuspensions with undigested tissues were filtered through a cellstrainer with pores of 40 μm (Millipore) and centrifuged. The cellpellets were resuspended in Dulbecco's Modified Eagle Medium NutrientMixture F-12 (DMEM/F12 (1:1) 1×, Gibco) supplemented with 10% fetalbovine serum and antibiotics penicillin (200 U/ml) and streptomycin (100U/mL). Cells were cultured at 37° C. in a humified atmosphere with 5%CO2 and were maintained in monolayer culture. In the experiment withantibiotic, 0.25 μg/mL clindamycin treatment was used.

After a few passages, cells were seeded in 6-well plates withoutantibiotics, allowed to attach overnight and assigned toPropionibacterium acnes treatment (ratio of 1:100 and 1:1000).Supplementation with LPS (200 ng/μl, Sigma-Aldrich) served as a positivecontrol, and cells cultured without P. acnes served as a negativecontrol. Cells were harvested into Qiazol lysis reagent (Qiagen) andRIPA buffer (Sigma) at four time points: after 3 h, 24 h, 48 h and 1week.

RNA Isolation

RNA was extracted by use of the Direct-zol RNA kit (Zymo Research) asdescribed in the manufacturer's instructions. The concentration andpurity of RNA were determined at 260 and 280 nm using a NanoDrop 2000(Thermo Scientific).

Quantitative Reverse Transcription Real-Time PCR (qRT-PCR)

The total amount of RNA was subjected to reverse transcription (RT) withthe use of High Capacity cDNA Reverse Transcription Kit (AppliedBiosystems). RT conditions were as follows: 10 min at 25° C., 2 h at 37°C. 5 min at 85° C. Real-time PCR was performed using TaqMan GeneExpression Master Mix (Applied Biosystems) and primer/probe systems:(Thermo Fisher): interleukin-1β (IL-1β, Hs01555410 ml), interleukin-1α(IL-1α, Hs00174092_m1), interleukin 6 (IL-6, Hs00174131_m1), interleukin8 (CXCL-8, Hs00174103_m1), chemokine (C—C motif) ligand 2 (CCL2,Hs00234140_m1), chemokine (C—C motif) ligand 3 (CCL3, HS00234142_m1),chemokine (C—C motif) ligand 4 (CCL4, Hs99999148_m1), nerve growthfactor (NGF, Hs00171458_m1), Brain-derived neurotrophic factor (BDNF,Hs02718934_s1).

All experiments were performed with glyceraldehyde phosphatedehydrogenase (GAPDH) as internal control using the real-timeQuantStudio 12K Flex system (Life Technologies). The fold-change of cDNAexpression levels was determined from the obtained ΔΔCt values comparedto ΔΔCt values of control samples. The thermal cycling amplificationprogram was as follows: one cycle of 94° C. for 10 min and 40 cycles of95° C. for 15 s, 60° C. for 60 s. Each sample was tested in triplicateand all real-time PCR reactions were run in duplicates.

Lipase Assay

Lipase activity was measured in cell-free culture supernatants collectedat 3 h, 24 h and 48 h time points. The procedure was performed usingLipase Activity Assay Kit II (MAK047, Sigma-Aldrich) according tomanufacturer protocol.

Enzyme-Linked ImmunoSorbent Assay (ELISA)

Cell lysates (RIPA, Sigma) were added in duplicates to ELISA plates,each with specific antibody against Human IL-beta (RAB0273A), Human IL-6(RAB0306), Human IL-8/CXCL8 (RABIL8A), Human MCP-1/CCL2 (RAB0054), HumanMIP-1 alpha/CCL3 (RAB0073), and Human MIP-1 beta/CCL-4 (RAB0075). Plateswere kept at 4° C. overnight and further proceeded according tomanufacturer protocol.

Data Normalization and Statistical Analyses

Student t-test was used for statistical evaluation of qRT-PCRexperiments (the values are given as the mean±SD). In all tests thevalue of p≤0.05 (*), the value of p≤0.01 (**) or the value of p≤0.001(***) was considered significant.

Example 2. Mechanism of Nociceptor Activation by P. acnes

Live bacteria actively release formylated peptides and secrete a host ofvirulence factors including pore-forming toxins (PFTs) to facilitatetissue dissemination (See Chiu et al, Nature 2013; 501:52-7).Alpha-hemolysin (Hla) is a PFT secreted by nearly all S. aureus strains,playing a role in tissue damage, bacterial spread, and inflammation andit was shown to be one of the mechanisms employed by S. aureus to elicitsustained bursts of calcium flux, selectively in capsaicin-responsiveneurons (See Chiu et al, Nature 2013; 501:52-7).

Type 1 secretion systems (T1SS) are wide-spread among Gram-negativebacteria. An important example is the secretion of the hemolytic toxinHlyA from uropathogenic strains. Secretion is achieved in a single stepdirectly from the cytosol to the extracellular space. The translocationmachinery is composed of three indispensable membrane proteins, two inthe inner membrane, and the third in the outer membrane. The innermembrane proteins belong to the ABC transporter and membrane fusionprotein families (MFPs), respectively, while the outer membranecomponent is a porin-like protein. Assembly of the three proteins istriggered by accumulation of the transport substrate (HlyA) in thecytoplasm, to form a continuous channel from the inner membrane,bridging the periplasm and finally to the exterior. Interestingly, themajority of substrates of T1SS contain all the information necessary fortargeting the polypeptide to the translocation channel—a specificsequence at the extreme C-terminus (See Thomas et al, Mol. Cell Res.2014; 1843:1629-41). From P. acnes, PPA1396 seems to resemble thisoperon structure more. It has also been annotated as hemolysin type A.

In bacteria, protein expression initiates with a formyl-methioninegroup, the formyl group is then removed post-translationally by peptidedeformylase. Formyl peptides can be released from the bacteria eitheractively or passively as a result of cell death. Neutrophils execute avariety of antimicrobial functions, including the generation of reactiveoxygen species (ROS), phagocytosis of pathogens and dead and dyingtissue, degranulation with the release of a variety of toxic products,expulsion of neutrophil extracellular traps, and paracrine signaling torecruit other cell types. Neutrophils sense inflammatory stimuliprincipally within the G protein-coupled receptor (GPCR) family. Thefirst GPCR to be described on the human neutrophil was formyl peptidereceptor 1 (FPR1) which, when activated, triggers a wide variety offunctions, including chemotaxis, degranulation, ROS production, andphagocytosis. The principal ligands for FPR1 are bacterial andmitochondrial formylated peptides, actively secreted by invadingpathogens or passively released from dead and dying host cells aftertissue injury.

Although it was initially thought that FPR1 only bound N-formylatedpeptides, it is now widely recognized that the formyl group is not aprerequisite for receptor binding. The N-formylated version of anypeptide containing a methionine residue at the 5′ terminus is at least100-fold more potent than the identical nonformylated peptide.

Several short formylated peptide sequences have been identified toelicit a response e.g. fMet-Leu-Phe (fMLF) by activating the formylpeptide receptors, and used routinely as models for the study of thesesystems. Various other peptides have been identified by studyingorganisms such as S. aureus (See Chiu et al, Nature 2013; 501:52-7) andL. monycogenes (See Rabiet et al, Eur. J. Immunol. 2005; 35:2486-95).

In this example, we show that a similar mechanism can exist in P. acnes,and which may contribute to chronic pain/inflammation.

Identification of P. acnes Formyl Peptides

P. acnes strains have a number of proteins that have the fMLF and fMLPpattern which has been identified in E. coli, but not fMIFL a patternderived from S. aureus. Nevertheless, as in other bacterial species itis expected that P. acnes release formylated peptides to itsenvironment, which can trigger an inflammatory response.

Among these proteins that have the peptides of interest there are twomembrane proteins and one secreted. Although these proteins are likelyto serve a different function in the cell, it is possible that they canbe released to the environment and elicit the neutrophil response.Similarly, a number of hypothetical proteins are detected in the genome,which may play a similar role, however they are not predicted to containany release mechanism.

TABLE 2 Predicted proteins with formyl peptide motif Genome FormylatedGene Locus Tag Gene Product Name Name peptide Symbol PPA1794glutamine--fructose-6-phosphate P. acnes MLF glmS transaminase KPA171202PPA0175 cytochrome bd-I ubiquinol oxidase P. acnes MLP PPA0175 Membranesubunit 2 apoprotein (EC 1.10.3.10) KPA171202 protein PPA1643D-serine/D-alanine/glycine:proton P. acnes MLP PPA1643 Membranesymporter, AAT family (TC 2.A.3.1.7) KPA171202 protein PPA0473 DNA orRNA helicase of superfamily II P. acnes MLP PPA0473 KPA171202 PPA0720DNA polymerase III, epsilon subunit P. acnes MLP PPA0720 (EC 2.7.7.7)KPA171202 PPA2183 hypothetical protein P. acnes MLP PPA2183 KPA171202PPA0854 hypothetical protein P. acnes MLP PPA0854 KPA171202 PPA0745hypothetical protein P. acnes MLP PPA0745 KPA171202 PPA2143 hypotheticalprotein (polysaccharide P. acnes MLP PPA2143 secreteddeacetylase/xylanase) KPA171202 protein PPA1734 nucleoside-bindingprotein P. acnes MLP PPA1734 KPA171202 PPA1371 putative transferase P.acnes MLP PPA1371 KPA171202 PPA0289 two component transcriptional P.acnes MLP PPA0289 regulator, LuxR family KPA171202

Identification of P. acnes Hemolysins

There are at least three proteins annotated as hemolysins in P. acnes,which are conserved in all sequenced genomes. Of the three, PPA1396 (andits orthologs) seem to better align to the operon structure of knownhemolysins. The gene is predicted to encode an alpha hemolysin, whichhas been shown to activate nociceptors (See Chiu et al, Nature 2013;501:52-7).

Notably, there is evidence, that the additional genes also exhibithemolytic activity.

TABLE 3 Predicted hemolysins Locus tag Annotation PPA0565 hemolysin IIIPPA0938 hemolysin PPA1396 alpha hemolysin

The three genes are present in all species/strains of Propionibacteriumacnes (aka Cutibacterium acnes).

TABLE 4 Hemolysis genes across P. acnes strains Genome Name PPA0938PPA1396 PPA0565 Cutibacterium acnes AE1 1 1 1 Cutibacterium acnes KCOM1861 1 1 1 Cutibacterium acnes PA_12_1_L1 1 1 1 Cutibacterium acnesPA_12_1_R1 1 1 1 Cutibacterium acnes PA_15_1_R1 1 1 1 Cutibacteriumacnes PA_15_2_L1 1 1 1 Cutibacterium acnes PA_21_1_L1 1 1 1Cutibacterium acnes PA_30_2_L1 1 1 1 Propionibacterium acnes 266 1 1 1Propionibacterium acnes 6609 1 1 1 Propionibacterium acnes ATCC 1 1 111828 Propionibacterium acnes C1 1 1 1 Propionibacterium acnes hdn-1 1 11 Propionibacterium acnes HL096PA1 1 1 1 Propionibacterium acnes 1 1 1KPA171202 Propionibacterium acnes SK137 1 1 1 Propionibacterium acnesTypeIA2 1 1 1 P. acn17 Propionibacterium acnes TypeIA2 1 1 1 P. acn31Propionibacterium acnes TypeIA2 1 1 1 P. acn33

Publications by Nodzo et al (Am. J. Orthop. Belle Mead N.J. 2014;43:E93-97) show that more than 50% of examined P. acnes strainsexhibited hemolytic activity, a population enriched in samples withdefinite infection. Since hemolysin is ubiquitous in P. acnes thisresult is somewhat unexpected—one would expect that all strains arehemolytic. However, it has been shown that non-hemolytic strains mayproduce hemolysis when treated with antibiotics or other stress (SeeWright et al, Infect. Dis. 2016; 9:39-44).

CONCLUSIONS

Based on bioinformatics analysis and correlation with S. aureus findingswe predict that P. acnes hemolysins play a role in causing chronic pain,and this prediction is supported by the presence of such genes in the P.acnes genome, as well as the evidence of their expression. This seems tobe a ubiquitous phenotype.

The potential of having formylated peptides playing this role is alsopresent. Since formylated peptides are ubiquitous among bacteria, and P.acnes contains at least a few proteins with sequence similar to peptidesthat have been shown to elicit response. However, it is possible thatother sequences can also play a similar role as it has been shown inother studies (See Rabiet et al, Eur. J. Immunol. 2005; 35:2486-95),either actively secreted by the bacterium or as byproducts of proteincleavage.

REFERENCES

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EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The invention claimed is:
 1. A method for treating degenerative discdisease or chronic lower back pain, comprising: administering to apatient having degenerative disc disease or chronic lower back pain, andsuspected of having a low-virulence infection, an antibiotic and anIL-1β inhibitor; wherein the IL-1β inhibitor is selected from the groupconsisting of anakinra, canakinumab, gevokizumab, and IL-1trap.
 2. Themethod of claim 1, wherein the patient is suspected of having alow-virulence infection based on the detection of a commensal pathogenin intervertebral disc tissue.
 3. The method of claim 2, wherein thecommensal pathogen is one or more of Propionibacterium sp.,Staphylococcus sp., Corynebacterium sp., Lactobacillus sp., Pseudomonassp., Enterococcus sp., Streptococcus sp., Bacillus sp., Citrobacter sp.,E. coli, Moraxella sp., Haemophilus sp., Neisseria sp., Clostridium sp.,Enterobacter sp., and Klebsiella sp.
 4. The method of claim 3, whereinthe commensal pathogen is Propionibacterium acnes.
 5. The method ofclaim 4, wherein the low-virulence infection is identified by thepresence of one or more virulence factors in the patient's disc tissue.6. The method of claim 1, wherein the antibiotic is selected frombenzylpenicillin, amoxicillin, ampicillin, dicloxacillin, methicillin,nafcillin, oxacillin, penicillin G, cephalexin, cefoxitin,cephalolothin, ceftriaxone, ciprofloxacin, chloramphenicol,erythromycin, tetracycline, vancomycin, clindamycin, fusidic acid,doxycycline, moxifloxacin, linezolid, rifampicin, ertapenem,taurolidine, or a combination thereof.
 7. The method of claim 1, whereinthe antibiotic is administered systemically or locally.
 8. The method ofclaim 7, wherein the antibiotic is administered by a route selected fromoral, parenteral, and transdermal.
 9. The method of claim 8, wherein theIL-1β antagonist is administered by a route selected from parenteral,oral, and transdermal.
 10. The method of claim 9, wherein the parenteraladministration is subcutaneous injection, intramuscular injection,intravenous injection, or local injection to intervertebral disc tissue.11. The method of claim 1, wherein the antibiotic and the IL-1βinhibitor are co-administered.
 12. The method of claim 1, wherein theantibiotic and the IL-1β inhibitor are administered prior to surgery.13. The method of claim 1, wherein the antibiotic and the IL-1βinhibitor are administered following surgery.